SOE PCR woes

: In article <Pine.SOL.3.91.960122181815.9403A-100000 at qlink>,
: Chanda Eva R <3erc at QLINK.QUEENSU.CA> wrote:
: >Dear netters:
: >I've tried several times to join 3 PCR fragments together by SOE, or
: >splicing by overlap extension (a.k.a. megapriming), but without success!
: >I followed the "standard" protocol (Horton et al,1993. Meth.Enzymol 217:
: >270-279)i.e. used 1/4 of a gel-purified, Genecleaned, 100uL PCR reaction of
: >each fragment, 1uM of each of the "outer" primers, 30 cycles of 1 min
: >@92C, 2 min @60C, 3 min @72C. My fragments overlap by 60 and 33 bp,
: >respectively, which is more than the protocol recommends--could that be
: >causing problems? (e.g. by annealing too slowly/inefficiently compared to
: >shorter overlaps) I'd GREATLY appreciate any practical tips anyone could
: >give me!!
: >Please save me from having to go back to old-fashioned splicing by
: >restriction-ligation subcloning!
: >Thanx,
: >Eva Chanda
: Drop your annealing temperature from 60C to 50C. The Horton paper recommends
: an annealing temperature of 50C. If that doesn't work, then try dropping the
: primer concentration by a factor of 10 as mentioned in another persons follow-
: up to your post.
: Speaking of woes about SOE PCR, I have been having intermittent luck with it
: by tring to do site directed mutagenesis in a gene. I produce two fragments,
: call them 5' and 3'. The 5' fragment is roughly 200bp and the 3' fragment is
: roughly 800bp. The overlap site is 18bp in length and the mutation site is
: in the 5' primer for the 3' fragment. The overlap sequence is:
: 5'-GTAGTCTAGAGACTTACC-3'
: The mutation site is approximately 10bp away from the end of this primer. I
: have been able to produce both fragments, but I do not seem to be able to pro-
: duce the overlap product. My PCR protocol calls for:
: 10X Reaction buffer : 10ul
: 2.5mM dNTP's : 8ul
: Outside primers (10uM concentration) : 1ul/primer
: Taq Polymerase: : 1ul (5 units/ul)
: For the reaction itself:
: Sterile, ddH2O : 69.5 ul
: 5' template : 1 ul (roughly 170ng/ul)
: 3' template : 1 ul (roughly 250ng/ul)
: MgCl2 (25 mM) : 8 ul
: reaction master mix (from above) : 20.5 ul
: Total volume: 100ul.
: The thermocylcer is set to run for 35 cycles with the following combinations:
: Cycle 1 : 5 min @ 94C
: Cycle 2-34 : 1 min @ 94C/2 min @ 50C/3 min @ 72C
: Cycle 35 : 10 min @ 72C
: I have been able to get this to work with one of the mutants that I am trying
: to produce (an Ala mutant), but the rest don't want to work. I have tried
: various ideas (different reaction buffers, lower template concentration, dif-
: ferent temperatures, etc). Is this what I can expect from SOE. Is it that
: flaky that some reactions will work and other won't? If you see anything that
: you might think I am doing wrong or you might think may be worthwhile, let me
: know. I am at my wits end with this one and my project has stalled because of
: it.
: Thanks in advance,
: Ido
You may be having problems with an untemplated A at the end of your PCR
product. I have been using BM's "expand" polymerase with this method &
it has been working great. These mixtures of Taq and a proofreading
polymerase supposedly will take off an untemplated A in these cases.
Strategene and BRL, among others make a similar product. You can usually beg
a sample out of them.
Good luck, hope this helps,
Mike Morales
Duke University